A longevity-specific bank of induced pluripotent stem cells from centenarians and their offspring.

Centenarians provide a unique lens through which to study longevity, healthy aging, and resiliency. Moreover, models of human aging and resilience to disease that allow for the testing of potential interventions are virtually non-existent. We obtained and characterized over 50 centenarian and offspring peripheral blood samples including those connected to functional independence data highlighting resistance to disability and cognitive impairment. Targeted methylation arrays were used in molecular aging clocks to compare and contrast differences between biological and chronological age in these specialized subjects. Isolated peripheral blood mononuclear cells (PBMCs) were then successfully reprogrammed into high-quality induced pluripotent stem cell (iPSC) lines which were functionally characterized for pluripotency, genomic stability, and the ability to undergo directed differentiation. The result of this work is a one-of-a-kind resource for studies of human longevity and resilience that can fuel the discovery and validation of novel therapeutics for aging-related disease.

Parasite manipulation of host phenotypes inferred from transcriptional analyses in a trematode-amphipod system.

Manipulation of host phenotypes by parasites is hypothesized to be an adaptive strategy enhancing parasite transmission across hosts and generations. Characterizing the molecular mechanisms of manipulation is important to advance our understanding of host-parasite coevolution. The trematode (Levinseniella byrdi) is known to alter the colour and behaviour of its amphipod host (Orchestia grillus) presumably increasing predation of amphipods which enhances trematode transmission through its life cycle. We sampled 24 infected and 24 uninfected amphipods from a salt marsh in Massachusetts to perform differential gene expression analysis. In addition, we constructed novel genomic tools for O. grillus including a de novo genome and transcriptome. We discovered that trematode infection results in upregulation of amphipod transcripts associated with pigmentation and detection of external stimuli, and downregulation of multiple amphipod transcripts implicated in invertebrate immune responses, such as vacuolar ATPase genes. We hypothesize that suppression of immune genes and the altered expression of genes associated with coloration and behaviour may allow the trematode to persist in the amphipod and engage in further biochemical manipulation that promotes transmission. The genomic tools and transcriptomic analyses reported provide new opportunities to discover how parasites alter diverse pathways underlying host phenotypic changes in natural populations.

Expression of retrotransposons contributes to aging in Drosophila.

Retrotransposons are a class of transposable elements capable of self-replication and insertion into new genomic locations. Across species, the mobilization of retrotransposons in somatic cells has been suggested to contribute to the cell and tissue functional decline that occurs during aging. Retrotransposons are broadly expressed across cell types, and de novo insertions have been observed to correlate with tumorigenesis. However, the extent to which new retrotransposon insertions occur during normal aging and their effect on cellular and animal function remains understudied. Here, we use a single nucleus whole genome sequencing approach in Drosophila to directly test whether transposon insertions increase with age in somatic cells. Analyses of nuclei from thoraces and indirect flight muscles using a newly developed pipeline, Retrofind, revealed no significant increase in the number of transposon insertions with age. Despite this, reducing the expression of two different retrotransposons, 412 and Roo, extended lifespan, but did not alter indicators of health such as stress resistance. This suggests a key role for transposon expression and not insertion in regulating longevity. Transcriptomic analyses revealed similar changes to gene expression in 412 and Roo knockdown flies and highlighted changes to genes involved in proteolysis and immune function as potential contributors to the observed changes in longevity. Combined, our data show a clear link between retrotransposon expression and aging.

Expression of Amyloidogenic Transthyretin Drives Hepatic Proteostasis Remodeling in an Induced Pluripotent Stem Cell Model of Systemic Amyloid Disease.

The systemic amyloidoses are diverse disorders in which misfolded proteins are secreted by effector organs and deposited as proteotoxic aggregates at downstream tissues. Although well described clinically, the contribution of synthesizing organs to amyloid disease pathogenesis is unknown. Here, we utilize hereditary transthyretin amyloidosis (ATTR amyloidosis) induced pluripotent stem cells (iPSCs) to define the contribution of hepatocyte-like cells (HLCs) to the proteotoxicity of secreted transthyretin (TTR). To this end, we generated isogenic, patient-specific iPSCs expressing either amyloidogenic or wild-type TTR. We combined this tool with single-cell RNA sequencing to identify hepatic proteostasis factors correlating with destabilized TTR production in iPSC-derived HLCs. By generating an ATF6 inducible patient-specific iPSC line, we demonstrated that enhancing hepatic ER proteostasis preferentially reduces the secretion of amyloidogenic TTR. These data highlight the liver's capacity to chaperone misfolded TTR prior to deposition, and moreover suggest the potential for unfolded protein response modulating therapeutics in the treatment of diverse systemic amyloidoses.

Author Correction: L1 drives IFN in senescent cells and promotes age-associated inflammation.

An Amendment to this paper has been published and can be accessed via a link at the top of the paper.

L1 drives IFN in senescent cells and promotes age-associated inflammation.

Retrotransposable elements are deleterious at many levels, and the failure of host surveillance systems for these elements can thus have negative consequences. However, the contribution of retrotransposon activity to ageing and age-associated diseases is not known. Here we show that during cellular senescence, L1 (also known as LINE-1) retrotransposable elements become transcriptionally derepressed and activate a type-I interferon (IFN-I) response. The IFN-I response is a phenotype of late senescence and contributes to the maintenance of the senescence-associated secretory phenotype. The IFN-I response is triggered by cytoplasmic L1 cDNA, and is antagonized by inhibitors of the L1 reverse transcriptase. Treatment of aged mice with the nucleoside reverse transcriptase inhibitor lamivudine downregulated IFN-I activation and age-associated inflammation (inflammaging) in several tissues. We propose that the activation of retrotransposons is an important component of sterile inflammation that is a hallmark of ageing, and that L1 reverse transcriptase is a relevant target for the treatment of age-associated disorders.

Modeling APC mutagenesis and familial adenomatous polyposis using human iPS cells.

Mutations in the gene Adenomatous Polyposis Coli or APC appear in most sporadic cases of colorectal cancer and it is the most frequent mutation causing hereditary Familial Adenomatous Polyposis. The detailed molecular mechanism by which APC mutations predispose to the development of colorectal cancer is not completely understood. This is in part due to the lack of accessibility to appropriate models that recapitulate the early events associated with APC mediated intestinal transformation. We have established a novel platform utilizing human induced Pluripotent Stem cells or iPSC from normal or FAP-specific APC mutant individuals and evaluated the effect of the mutation in the cells before and after differentiation into intestinal organoids. In order to minimize genetic background effects, we also established an isogenic platform using TALEN-mediated gene editing. Comparison of normal and APC mutant iPSC revealed a significant defect in cell identity and polarity due to the presence of APC in heterozygosity as well as chromosomal aberrations including abnormal anaphases and centrosome numbers. Importantly, upon specification into intestinal progeny, APC heterozygosity was responsible for a major change in the transcriptional identity of the cells with dysregulation of key signaling pathways, including metabolic reprogramming, abnormal lipid metabolism and intestinal-specific cadherin expression. In conclusion, we have developed a novel iPSC/intestinal model of APC mutagenesis and provide strong evidence that APC in heterozygosity imparts a clear phenotypic and molecular defect, affecting basic cellular functions and integrity, providing novel insights in the earlier events of APC-mediated tumorigenesis.

Notch and Aryl Hydrocarbon Receptor Signaling Impact Definitive Hematopoiesis from Human Pluripotent Stem Cells.

Induced pluripotent stem cells (iPSCs) stand to revolutionize the way we study human development, model disease, and eventually, treat patients. However, these cell sources produce progeny that retain embryonic and/or fetal characteristics. The failure to mature to definitive, adult-type cells is a major barrier for iPSC-based disease modeling and drug discovery. To directly address these concerns, we have developed a chemically defined, serum and feeder-free-directed differentiation platform to generate hematopoietic stem-progenitor cells (HSPCs) and resultant adult-type progeny from iPSCs. This system allows for strict control of signaling pathways over time through growth factor and/or small molecule modulation. Through direct comparison with our previously described protocol for the production of primitive wave hematopoietic cells, we demonstrate that induced HSPCs are enhanced for erythroid and myeloid colony forming potential, and strikingly, resultant erythroid-lineage cells display enhanced expression of adult ß globin indicating definitive pathway patterning. Using this system, we demonstrate the stage-specific roles of two key signaling pathways, Notch and the aryl hydrocarbon receptor (AHR), in the derivation of definitive hematopoietic cells. We illustrate the stage-specific necessity of Notch signaling in the emergence of hematopoietic progenitors and downstream definitive, adult-type erythroblasts. We also show that genetic or small molecule inhibition of the AHR results in the increased production of CD34+ CD45+ HSPCs while conversely, activation of the same receptor results in a block of hematopoietic cell emergence. Results presented here should have broad implications for hematopoietic stem cell transplantation and future clinical translation of iPSC-derived blood cells. Stem Cells 2018;36:1004-1019.

Prospective isolation of NKX2-1-expressing human lung progenitors derived from pluripotent stem cells.

It has been postulated that during human fetal development, all cells of the lung epithelium derive from embryonic, endodermal, NK2 homeobox 1-expressing (NKX2-1+) precursor cells. However, this hypothesis has not been formally tested owing to an inability to purify or track these progenitors for detailed characterization. Here we have engineered and developmentally differentiated NKX2-1GFP reporter pluripotent stem cells (PSCs) in vitro to generate and isolate human primordial lung progenitors that express NKX2-1 but are initially devoid of differentiated lung lineage markers. After sorting to purity, these primordial lung progenitors exhibited lung epithelial maturation. In the absence of mesenchymal coculture support, this NKX2-1+ population was able to generate epithelial-only spheroids in defined 3D cultures. Alternatively, when recombined with fetal mouse lung mesenchyme, the cells recapitulated epithelial-mesenchymal developing lung interactions. We imaged these progenitors in real time and performed time-series global transcriptomic profiling and single-cell RNA sequencing as they moved through the earliest moments of lung lineage specification. The profiles indicated that evolutionarily conserved, stage-dependent gene signatures of early lung development are expressed in primordial human lung progenitors and revealed a CD47hiCD26lo cell surface phenotype that allows their prospective isolation from untargeted, patient-specific PSCs for further in vitro differentiation and future applications in regenerative medicine.